TNFalpha antagonists and methotrexate in the treatment of TNF-mediated disease

ABSTRACT

Methods for treating and/or preventing a TNF-mediated disease in an individual are disclosed. Also disclosed is a composition comprising methotrexate and an anti-tumor necrosis factor antibody. TNF-mediated diseases include rheumatoid arthritis, Crohn&#39;s disease, and acute and chronic immune diseases associated with transplantation.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.08/690,775, filed Aug. 1, 1996, which is a continuation-in-part of U.S.application Ser. No. 08/607,419, filed Feb. 28, 1996, now abandoned,which is a continuation-in-part of International Application No.PCT/GB94/00462, filed Mar. 10, 1994, which is a continuation-in-part ofU.S. application Ser. No. 08/403,785, now U.S. Pat. No. 5,741,488, whichis the U.S. National Phase of International Application No.PCT/GB93/02070, filed Oct. 6, 1993, which is a continuation-in-part ofU.S. application Ser. No. 07/958,248, filed Oct. 8, 1992, now abandoned,the teachings of all of which are entirely incorporated herein byreference.

BACKGROUND OF THE INVENTION

Monocytes and macrophages secrete cytokines known as tumor necrosisfactor alpha (TNFα) and tumor necrosis factor beta (TNFβ) in response toendotoxin or other stimuli. TNFα is a soluble homotrimer of 17 kDprotein subunits (Smith et al., J. Biol. Chem. 262:6951-6954 (1987)). Amembrane-bound 26 kD precursor form of TNF also exists (Kriegler et al.,Cell 53:45-53 (1988)). For reviews of TNF, see Beutler et al., Nature320:584 (1986); Old, Science 230:630 (1986); and Le et al., Lab. Invest.56:234 (1987).

Cells other than monocytes or macrophages also produce TNFα. Forexample, human non-monocytic tumor cell lines produce tumor necrosisfactor (TNF) (Rubin et al., J. Exp. Med. 164:1350 (1986); Spriggs etal., Proc. Natl. Acad. Sci. USA 84:6563 (1987)). CD4+ and CD8+peripheral blood T lymphocytes and some cultured T and B cell lines(Cuturi et al., J. Exp. Med. 165:1581 (1987); Sunig et al., J. Exp. Med.168:1539 (1988); Turner et al., Eur. J. Immunol. 17:1807-1814 (1987))also produce TNFα.

TNF causes pro-inflammatory actions which result in tissue injury, suchas degradation of cartilage and bone, induction of adhesion molecules,inducing procoagulant activity on vascular endothelial cells (Pober etal., J. Immunol. 136:1680 (1986)), increasing the adherence ofneutrophils and lymphocytes (Pober et al., J. Immunol. 138:3319 (1987)),and stimulating the release of platelet activating factor frommacrophages, neutrophils and vascular endothelial cells (Camussi et al.,J. Exp. Med. 166: 1390 (1987)).

Recent evidence associates TNF with infections (Cerami et al., Immunol.Today 9:28 (1988)), immune disorders, neoplastic pathologies (Oliff etal., Cell 50:555 (1987)), autoimmune pathologies and graft-versus-hostpathologies (Piguet et al., J. Exp. Med. 166:1280 (1987)). Theassociation of TNF with cancer and infectious pathologies is oftenrelated to the host's catabolic state. Cancer patients suffer fromweight loss, usually associated with anorexia.

The extensive wasting which is associated with cancer, and otherdiseases, is known as “cachexia” (Kern et al., J. Parent. Enter. Nutr.12:286-298 (1988)). Cachexia includes progressive weight loss, anorexia,and persistent erosion of body mass in response to a malignant growth.The fundamental physiological derangement can relate to a decline infood intake relative to energy expenditure. The cachectic state causesmost cancer morbidity and mortality. TNF can mediate cachexia in cancer,infectious pathology, and other catabolic states.

TNF also plays a central role in gram-negative sepsis and endotoxicshock (Michie et al., Br. J. Surg. 76:670-671 (1989); Debets et al.,Second Vienna Shock Forum, p. 463-466 (1989); Simpson et al., Crit. CareClin. 5:27-47 (1989)), including fever, malaise, anorexia, and cachexia.Endotoxin strongly activates monocyte/macrophage production andsecretion of TNF and other cytokines (Kombluth et al., J. Immunol.137:2585-2591 (1986)). TNF and other monocyte-derived cytokines mediatethe metabolic and neurohormonal responses to endotoxin (Michie et al.,New Engl. J. Med. 318:1481-1486 (1988)). Endotoxin administration tohuman volunteers produces acute illness with flu-like symptoms includingfever, tachycardia, increased metabolic rate and stress hormone release(Revhaug et al., Arch. Surg. 123:162-170 (1988)). Circulating TNFincreases in patients suffering from Gram-negative sepsis (Waage et al.,Lancet 1:355-357 (1987); Hammerle et al., Second Vienna Shock Forum p.715-718 (1989); Debets et al., Crit. Care Med. 17:489-497 (1989);Calandra et al., J. Infect. Dis. 161:982-987 (1990)).

Thus, TNFα has been implicated in inflammatory diseases, autoimmunediseases, viral, bacterial and parasitic infections, malignancies,and/or neurogenerative diseases and is a useful target for specificbiological therapy in diseases, such as rheumatoid arthritis andCropl-'s disease. Beneficial effects in open-label trials with achimeric monoclonal antibody to TNFα (cA2) have been reported withsuppression of inflammation (Elliott et al., Arthritis Rheum.36:1681-1690 (1993); Elliott et al., Lancet 344:1125-1127 (1994)). Seealso, Van Dullemeen et al., Gastroenterology 109:129-135 (1995).Beneficial results in a randomized, double-blind, placebo-controlledtrial with cA2 have also been reported with suppression of inflammation(Elliott et al., Lancet 344:1105-1110 (1994)).

SUMMARY OF THE INVENTION

The present invention is based on the discovery that treatment ofpatients suffering from a TNF-mediated disease with a tumor necrosisfactor antagonist, Such as an anti-tumor necrosis factor antibody, asadjunctive and/or concomitant therapy to methotrexate therapy produces arapid and sustained reduction in the clinical signs and symptoms of thedisease. The present invention is also based on the unexpected anddramatic discovery that a multiple dose regimen of a tumor necrosisfactor antagonist, such as an anti-tumor necrosis factor antibody, whenadministered adjunctively with methotrexate to an individual sufferingfrom a TNF-mediated disease produces a highly beneficial or synergisticclinical response for a significantly longer duration compared to thatobtained with a single or multiple dose regimen of the antagonistadministered alone or that obtained with methotrexate administeredalone. As a result of Applicants' invention, a method is provided hereinfor treating and/or preventing a TNF-mediated disease in an individualcomprising co-administering an anti-TNF antibody or a fragment thereofand methotrexate to the individual in therapeutically effective amounts.In a particular embodiment, methotrexate is administered in the form ofa series of low doses separated by intervals of days or weeks.

A method is also provided herein for treating and/or preventingrecurrence of a TNF-mediated disease in an individual comprisingco-administering an anti-TNT antibody or a fragment thereof andmethotrexate to the individual in therapeutically effective amounts.TNF-mediated diseases include rheumatoid arthritis, Crolh's disease, andacute and chronic immune diseases associated with an allogenictransplantation (e.g., renal, cardiac, bone marrow, liver, pancreatic,small intestine, skin or lung transplantation).

Therefore, in one embodiment, the invention relates to a method oftreating and/or preventing rheumatoid arthritis in an individualcomprising co-administering an anti-TNF antibody or a fragment thereofand methotrexate to the individual in therapeutically effective amounts.In a second embodiment, the invention relates to a method of treatingand/or preventing Croli's disease in an individual comprisingco-administering an anti-TNF antibody or a fragment thereof andmethotrexate to the individual in therapeutically effective amounts. Ina third embodiment, the invention relates to a method of treating and/orpreventing other autoimmune diseases and/or acute or chronic immunedisease associated with a transplantation in an individual, comprisingco-administering an anti-TNF antibody or a fragment thereof andmethotrexate to the individual in therapeutically effective amounts.

A further embodiment of the invention relates to compositions comprisingan anti-TNF antibody or a fragment thereof and methotrexate.

In addition to anti-TNF antibodies, TNF antagonists include anti-TNFantibodies and receptor molecules which bind specifically to TNF;compounds which prevent and/or inhibit TNF synthesis, TNF release or itsaction on target cells, such as thalidomide, tenidap, phosphodiesteraseinhibitors (e.g, pentoxifylline and rolipram), A2b adenosine receptoragonists and A2b adenosine receptor enhancers; and compounds whichprevent and/or inhibit TNF receptor signalling.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C are a set of three graphs showing the results over time forswollen joint count in rheumatoid arthritis (RA) patients receiving cA2treatment (1 mg/kg, 3 mg/kg or 10 mg/kg) with or without methotrexate.Results for the placebo group (methotrexate alone) are shown with the 1mg/kg group. The number of patients with data at each evaluation visitis shown at the bottom of each graph. White circle=−methotrexate (MTX−);black circle=+methotrexate (MTX+); square=placebo.

FIGS. 2A-2C are a set of three graphs showing the results over time fortender joint count in RA patients receiving cA2 treatment (1 mg/kg, 3mg/kg or 10 mg/kg) with or without methotrexate. Results for the placebogroup (methotrexate alone) are shown with the 1 mg/kg group. The numberof patients with data at each evaluation visit is shown at the bottom ofeach graph. White circle=−methotrexate; black circle+methotrexate;square=placebo.

FIGS. 3A-3C are a set of three graphs showing the results over time forthe Physician's Global Disease Assessment in RA patients receiving cA2treatment (1 mg/kg, 3 mg/kg or 10 mg/kg) with or without methotrexate.Results for the placebo group (methotrexate alone) are shown with the 1mg/kg group. The number of patients with data at each evaluation visitis shown at the bottom of each graph. White circle=−methotrexate; blackcircle=+methotrexate; square=placebo.

FIGS. 4A-4C are a set of three graphs showing the results over time forthe Patient, Disease Assessment in RA patients receiving cA2 treatment(1 mg/kg, 3 mg/kg or 10 mg/kg) with or without methotrexate. Results forthe placebo group (methotrexate alone) are shown with the 1 mg/kg group.The number of patients with data at each evaluation visit is shown atthe bottom of each graph. White circle=−methotrexate; blackcircle=+methotrexate; square=placebo.

FIGS. 5A-5C are a set of three graphs showing the results over time forC-reactive protein (CRP) concentration in RA patients receiving cA2treatment (1 mg/cg, 3 mg/kg or 10 mg/kg) with or without methotrexate.Results for the placebo group (methotrexate alone) are shown with the 1mg/kg group. The number of patients with data at each evaluation visitis shown at the bottom of each graph. White circle=−methotrexate; blackcircle=+methotrexate; square=placebo.

FIGS. 6A-6C are a set of three graphs showing the results over time forthe Health Assessment Questionnaire (HAQ) in RA patients receiving cA2treatment (1 mg/kg, 3 mg/kg or 10 mg/kg) with or without methotrexate.Results for the placebo group (methotrexate alone) are shown with the 1mg/kg group. The number of patients with data at each evaluation visitis shown at the bottom of each graph. White circle=−methotrexate; blackcircle=+methotrexate; square=placebo.

FIGS. 7A-7F are a set of six graphs showing the serum cA2 concentrationin each RA patient receiving cA2 treatment (1 mg/kg, 3 mg/kg or 10mg/kg) with or without methotrexate, plotted over time. Data plotted arethe serum cA2 concentrations obtained just before the administration ofcA2 at weeks 2, 6, 10 and 14 and then at weeks 18 and 26. The scales forthe serum cA2 concentration are condensed with higher doses of cA2.

FIGS. 8A and 8B are a set of two graphs showing the median serum cA2concentration over time in RA patients receiving 3 mg/kg cA2 (top panel)or 10 mg/kg cA2 (bottom panel) with or without methotrexate.Square=+methotrexate; circle or triangle=−methotrexate.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the discovery that tumor necrosisfactor antagonists can be administered to patients suffering from aTNF-mediated disease as adjunctive and/or concomitant therapy tomethotrexate therapy, with good to excellent alleviation of the signsand symptoms of the disease. The present invention also relates to thediscovery that tumor necrosis factor antagonists can be administered topatients suffering from a TNF-mediated disease in multiple doses and asadjunctive and/or concomitant therapy to methotrexate therapy, with asignificant improvement in duration of clinical response.

As a result of Applicants' invention, a method is provided herein fortreating and/or preventing a TNF-mediated disease in an individual,comprising co-administering methotrexate and a tumor necrosis factorantagonist to the individual in therapeutically effective amounts. TheTNF antagonist and methotrexate can be administered simultaneously orsequentially. The TNF antagonist and methotrexate can each beadministered in single or multiple doses. Multiple TNF antagonists canbe co-administered with methotrexate. Other therapeutic regimens andagents can be used in combination with the therapeutic co-administrationof TNF antagonists and methotrexate or other drugs that suppress theimmune system.

A method is also provided herein for treating and/or preventingrecurrence of a TNF-mediated disease in an individual comprisingco-administering methotrexate and a TNF antagonist to the individual intherapeutically effective amounts.

As used herein, a “TNF-mediated disease” refers to a TNF relatedpathology or disease. TNF related pathologies or diseases include, butare not limited to, the following:

(A) acute and chronic immune and autoimmune pathologies, such as, butnot limited to, rheumatoid arthritis (RA), juvenile chronic arthritis(JCA), thyroiditis, graft versus host disease (GVHD), scleroderma,diabetes mellitus, Graves' disease, allergy acute or chronic immunedisease associated with an allogenic transplantation, such as, but notlimited to, renal transplantation, cardiac transplantation, bone marrowtransplantation, liver transplantation, pancreatic transplantation,small intestine transplantation, lung transplantation and skintransplantation;

(B) infections, including, but not limited to, sepsis syndrome,cachexia, circulatory collapse and shock resulting from acute or chronicbacterial infection, acute and chronic parasitic and/or infectiousdiseases, bacterial, viral or fungal, such as a human immunodeficiencyvirus (HIV), acquired immunodeficiency syndrome (AIDS) (includingsymptoms of cachexia, autoimmune disorders, AIDS dementia complex andinfections);

(C) inflammatory diseases, such as chronic inflammatory pathologies,including chronic inflammatory pathologies such as, but not limited to,sarcoidosis, chronic inflammatory bowel disease, ulcerative colitis, andCrohn's pathology or disease; vascular inflammatory pathologies, suchas, but not limited to, disseminated intravascular coagulation,atherosclerosis, Kawasaki's pathology and vasculitis syndromes, such as,but not limited to, polyarteritis nodosa, Wegener's granulomatosis,Henoch-Schönlein purpura, giant cell arthritis and microscopicvasculitis of the kidneys; chronic active hepatitis; Sjögren's syndrome;spondyloarthropathies, such as ankylosing spondylitis, psoriaticarthritis and sponidylitis, enteropathic arthritis and spondylitis,reactive arthritis and arthritis associated with inflammatory boweldisease; and uveitis;

(D) neurodegenerative diseases, including, but not limited to,demyelinating diseases, such as multiple sclerosis and acute transversemyelitis; myasthenia gravis; extrapyramidal and cerebellar disorders,such as lesions of the corticospinal system; disorders of the basalganglia or cerebellar disorders; hyperkinetic movement disorders, suchas Huntington's chorea and senile chorea; drug-induced movementdisorders, such as those induced by drugs which block central nervoussystem (CNS) dopamine receptors; hypokinetic movement disorders, such asParkinson's disease; progressive supranuclear palsy; cerebellar andspinocerebellar disorders, such as astructural lesions of thecerebellum; spinocerebellar degenerations (spinal ataxia, Friedreich'sataxia, cerebellar cortical degenerations, multiple systemsdegenerations (Mencel, Dejerine-Thomas, Shi-Drager, and MachadoJoseph));and systemic disorders (Refsurn's disease, abetalipoproteinemia, ataxia,telangiectasia, and mitochondrial multisystem disorder); disorders ofthe motor unit, such as neurogenic muscular atrophies (anterior horncell degeneration, such as amyotrophic lateral sclerosis, infantilespinal muscular atrophy and juvenile spinal muscular atrophy);Alzheimer's disease; Down's syndrome in middle age; diffuse Lewy bodydisease; senile dementia of Lewy body type; Wernicke-Korsakoff syndrome;chronic alcoholism; primary biliary cirrhosis; cryptogenic fibrosingalveolitis and other fibrotic lung diseases; hemolytic anemia;Creutzfeldt-Jakob disease; sub acute sclerosing panencephalitis,Hallervorden-Spatz disease; and dementia pugilistica, or any subsetthereof;

(E) malignant pathologies involving TNF-secreting tumors or othermalignancies involving TNF, such as, but not limited to, leukemias(acute, chronic myelocytic, chronic lymphocytic and/or myelodyspasticsyndrome); lymphomas (Hodgkin's and non-Hodgkin's lymphomas, such asmalignant lymphomas (Burkitt's lymphoma or Mycosis fungoides));

(F) cachectic syndromes and other pathologies and diseases involvingexcess TNF, such as, but not limited to, cachexia of cancer, parasiticdisease and heart failure; and

(G) alcohol-induced hepatitis and other forms of chronic hepatitis.

See, e.g., Berkow et al., Eds., The Merck Manual, 16th edition, chapter11, pp. 1380-1529, Merck and Co., Rahway, N.J., 1992, incorporatedherein by reference.

The terms “recurrence”, “flare-up” or “relapse” are defined to encompassthe reappearance of one or more symptoms of the disease state. Forexample, in the case of rheumatoid arthritis, a reoccurrence can includethe experience of one or more of swollen joints, morning stiffness orjoint tenderness.

In one embodiment, the invention relates to a method of treating and/orpreventing rheumatoid arthritis in an individual comprisingco-administering methotrexate and a TNF antagonist to the individual intherapeutically effective amounts.

In a second embodiment, the invention relates to a method for treatingand/or preventing Croln's disease in an individual comprisingco-administering a methotrexate and a TNF antagonist to the individualin therapeutically effective amounts.

In a third embodiment, the invention relates to a method for treatingand/or preventing an acute or chronic immune disease associated with anallogenic transplantation in an individual comprising co-administeringmethotrexate and a TNF antagonist to the individual in therapeuticallyeffective amounts. As used herein, a “transplantation” includes organ,tissue or cell transplantation, such as renal transplantation, cardiactransplantation, bone marrow transplantation, liver transplantation,pancreatic transplantation, small intestine transplantation, skintransplantation and lung transplantation.

The benefits of combination therapy with methotrexate and TNFantagonists include high clinical response rates for significantlylonger durations in comparison with that obtained with treatment witheach therapeutic modality separately. In addition, methotrexatesignificantly reduces immunogenicity of anti-TNF antibodies, thuspermitting administration of multiple dosages of anti-TNF antibodieswith enhanced safety. The results described herein suggest thatmethotrexate can be used to reduce immunogenicity of other antibodies orproteins. Based on the results described herein, methotrexate can beused in other forms of antibody therapy, such as anti-IL-2 antibodytherapy. This method is particularly pertinent in therapies other thananti-CD4 antibody therapy.

In a further embodiment, the invention relates to compositionscomprising methotrexate and a TNF antagonist. The compositions of thepresent invention are useful for treating a subject having a pathologyor condition associated with abnormal levels of a substance reactivewith a TNF antagonist, in particular TNF in excess of, or less than,levels present in a normal healthy subject, where such excess ordiminished levels occur in a systemic, localized or particular tissuetype or location in the body. Such tissue types can include, but are notlimited to, blood, lymph, central nervous system (CNS), liver, kidney,spleen, heart muscle or blood vessels, brain or spinal cord white matteror grey matter, cartilage, ligaments, tendons, lung, pancreas, ovary,testes, prostate. Increased or decreased TNF concentrations relative tonormal levels can also be localized to specific regions or cells in thebody, such as joints, nerve blood vessel junctions, bones, specifictendons or ligaments, or sites of infection, such as bacterial or viralinfections.

Tumor Necrosis Factor Antagonists

As used herein, a “tumor necrosis factor antagonist” decreases, blocks,inhibits, abrogates or interferes with TNF activity in vivo. Forexample, a suitable TNF antagonist can bind TNF and includes anti-TNFantibodies and receptor molecules which bind specifically to TNF. Asuitable TNF antagonist can also prevent or inhibit TNF synthesis and/orTNF release and includes compounds such as thalidomide, tenidap, andphosphodiesterase inhibitors, such as, but not limited to,pentoxifylline and rolipram. A suitable TNT antagonist that can preventor inhibit TNF synthesis and/or TNF release also includes A2b adenosinereceptor enhancers and A2b adenosine receptor agonists (e.g.,5′-(N-cyclopropyl)-carboxamidoadenosine, 5′-N-ethylcarboxamidoadenosine,cyclohexyladenosine and R-N⁶-phenyl-2-propyladenosine). See, forexample, Jacobson (GB 2 289 218 A), the teachings of which are entirelyincorporated herein by reference. A suitable TNF antagonist can alsoprevent or inhibit TNF receptor signalling.

Anti-TNF Antibodies

As used herein, an “anti-tumor necrosis factor antibody” decreases,blocks, inhibits, abrogates or interferes with TNF activity in vivo.Anti-TNF antibodies useful in the methods and compositions of thepresent invention include monoclonal, chimeric, humanized, resurfacedand recombinant antibodies and fragments thereof which are characterizedby high affinity binding to TNF and low toxicity (including humananti-murine antibody (HAMA) and/or human anti-chimeric antibody (HACA)response). In particular, an antibody where the individual components,such as the variable region, constant region and framework, individuallyand/or collectively possess low immunogenicity is useful in the presentinvention. The antibodies which can be used in the invention arecharacterized by their ability to treat patients for extended periodswith good to excellent alleviation of symptoms and low toxicity. Lowimmunogenicity and/or high affinity, as well as other undefinedproperties, may contribute to the therapeutic results achieved.

An example of a high affinity monoclonal antibody useful in the methodsand compositions of the present invention is murine monoclonal antibody(mAb) A2 and antibodies which will competitively inhibit in vivo thebinding to human TNFα of anti-TNFα murine mAb A2 or an antibody havingsubstantially the same specific binding characteristics, as well asfragments and regions thereof. Murine monoclonal antibody A2 andchimeric derivatives thereof, such as cA2, are described in U.S.application Ser. No. 08/192,093 (filed Feb. 4, 1994), U.S. applicationSer. No. 08/192,102 (filed Feb. 4, 1994; now U.S. Pat. No. 5,656,272),U.S. application Ser. No. 08/192,861 (filed Feb. 4, 1994; now U.S. Pat.No. 5,919,452), U.S. application Ser. No. 08/324,799 (filed Oct. 18,1994; now U.S. Pat. No. 5,698,195), and Le, J. et al., InternationalPublication No. WO 92/16553 (published Oct. 1, 1992), which referencesare entirely incorporated herein by reference. A second example of ahigh affinity monoclonal antibody useful in the methods and compositionsof the present invention is murine mAb 195 and antibodies which willcompetitively inhibit in vivo the binding to human TNFα of anti-TNTαmurine 195 or an antibody having substantially the same specific bindingcharacteristics, as well as fragments and regions thereof. Other highaffinity monoclonal antibodies useful in the methods and compositions ofthe present invention include murine mAb 114 and murine mAb 199 andantibodies which will competitively inhibit in vivo the binding to humanTNFα of anti-TNFα murine mAb 114 or mAb 199 or an antibody havingsubstantially the same specific binding characteristics of mAb 114 ormAb 199, as well as fragments and regions thereof. Murine monoclonalantibodies 114, 195 and 199 and the method for producing them aredescribed by Möller, A. et al. (Cytokine 2(3):162-169 (1990)), theteachings of which are entirely incorporated herein by reference.Preferred methods for determining mAb specificity and affinity bycompetitive inhibition can be found in Harlow, et al., Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1988); Colligan et al., eds., Current Protocols inImmunology, Greene Publishing Assoc. and Wiley Interscience, New York(1992, 1993); Kozbor et al., Immunol. Today 4:72-79 (1983); Ausubel etal., eds., Current Protocols in Molecular Biology, Wiley Interscience,New York (1987, 1992, 1993); and Muller, Meth. Enzymol. 92:589-601(1983), which references are entirely incorporated herein by reference.

Additional examples of monoclonal anti-TNF antibodies that can be usedin the present invention are described in the art (see, e.g., U.S.application Ser. No. 07/943,852 (filed Sep. 11, 1992); Rathjen et al.,International Publication No. WO 91/02078 (published Feb. 21, 1991);Rubin et al., EPO Patent Publication 0218868 (published Apr. 22, 1987);Yone et al., EPO Patent Publication No. 0288088 (Oct. 26, 1988); Liang,et al., Biochem. Biophys. Res. Comm. 137:847-854 (1986); Meager, et al.,Hybridoma 6:305-311 (1987); Fendly et al., Hybridoma 6:359-369 (1987);Bringman, et al., Hybridoma 6:489-507 (1987); Hirai, et al., J. Immuno.Meth. 96:57-62 (1987); Moller, et al., Cytokine 2:162-169 (1990), whichreferences are entirely incorporated herein by reference).

Chimeric antibodies are immunoglobulin molecules characterized by two ormore segments or portions derived from different animal species.Generally, the variable region of the chimeric antibody is derived froma non-human mammalian antibody, such as a murine mAb, and theimmunoglobulin constant region is derived from a human immunoglobulinmolecule. Preferably, a variable region with low immunogenicity isselected and combined with a human constant region which also has lowimmunogenicity, the combination also preferably having lowimmunogenicity. “Low” immunogenicity is defined herein as raisingsignificant HACA or HAMA responses in less than about 75%, or preferablyless than about 50% of the patients treated and/or raising low titres inthe patient treated (less than about 300, preferably less than about 100measured with a double antigen enzyme immunoassay) (Elliott et al.,Lancet 344:1125-1127 (1994), incorporated herein by reference).

As used herein, the term “chimeric antibody” includes monovalent,divalent or polyvalent immunoglobulins. A monovalent chimeric antibodyis a dimer (HL)) formed by a chimeric H chain associated throughdisulfide bridges with a chimeric L chain. A divalent chimeric antibodyis a tetramer (H2L2) formed by two HL dimers associated through at leastone disulfide bridge. A polyvalent chimeric antibody can also beproduced, for example, by employing a CH region that aggregates (e.g.,from an IgM H chain, or μ chain).

Antibodies comprise individual heavy (H) and/or light (L) immunoglobulinchains. A chimeric H chain comprises an antigen binding region derivedfrom the H chain of a non-human antibody specific for TNF, which islinked to at least a portion of a human H chain C region (CH), such asCH1 or CH2. A chimeric L chain comprises an antigen binding regionderived from the L chain of a non-human antibody specific for TNF,linked to at least a portion of a human L chain C region (CL).

Chimeric antibodies and methods for their production have been describedin the art (Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855(1984); Boulianine et al., Nature 312:643-646 (1984); Neuberger et al.,Nature 314:268-270 (1985); Taniguchi et al., European Patent ApplicationNo. 171496 (published Feb. 19, 1985); Morrison et al., European PatentApplication No. 173494 (published Mar. 5, 1986); Neuberger et al., PCTApplication No. WO 86/01533, (published Mar. 13, 1986); Kudo et al.,European Patent Application No. 184187 (published Jun. 11, 1986);Morrison et al., European Patent Application No. 173494 (published Mar.5, 1986); Sahagan et al., J. Immunol. 137:1066-1074 (1986); Robinson etal., International Publication No. PCT/US86/02269 (published May 7,1987); Liu et al., Proc. Natl. Acad. Sci. USA 84:3439-3443 (1987); Sunet al., Proc. Natl. Acad. Sci. USA 84:214-218 (1987); Better et al.,Science 240:1041-1043 (1988); and Harlow and Lane, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory, New York, 1988). Thesereferences are entirely incorporated herein by reference.

The anti-TNF chimeric antibody can comprise, for example, two lightchains and two heavy chains, each of the chains comprising at least partof a human constaint region and at least part of a variable (V) regionof non-human origin having specificity to human TNF, said antibodybinding with high affinity to an inhibiting and/or neutralizing epitopeof human TNF, such as the antibody cA2. The antibody also includes afragment or a derivative of such an antibody, such as one or moreportions of the antibody chain, such as the heavy chain constant orvariable regions, or the light chain constant or variable regions.

Humanizing and resurfacing the antibody can further reduce theimmunogenicity of the antibody. See, for example, Winter (U.S. Pat. No.5,225,539 and EP 239,400 B1), Padlan et al. (EP 519,596 A1) and Pedersenet al. (EP 592,106 A1). These references are incorporated herein byreference.

Preferred antibodies useful in the methods and compositions of thepresent invention are high affinity human-murine chimeric anti-TNFantibodies, and fragments or regions thereof, that have potentinhibiting and/or neutralizing activity in vivo against human TNFα. Suchantibodies and chimeric antibodies can include those generated byimmunization using purified recombinant TNFα or peptide fragmentsthereof comprising one or more epitopes.

An example of such a chimeric antibody is cA2 and antibodies which willcompetitively inhibit in vivo the binding to human TNFα of anti-TNFαmurine mAb A2, chimeric mAb cA2, or an antibody having substantially thesame specific binding characteristics, as well as fragments and regionsthereof. Chimeric mAb cA2 has been described, for example, in U.S.application Ser. No. 08/192,093 (filed Feb. 4, 1994), U.S. applicationSer. No. 08/192,102 (filed Feb. 4, 1994; now U.S. Pat. No. 5,656,272),U.S. application Ser. No. 08/192,861 (filed Feb. 4, 1994; now U.S. Pat.No. 5,919,452), and U.S. application Ser. No. 08/324,799 (filed Oct. 18,1994; now U.S. Pat. No. 5,698,195), and by Le, J. et al. (InternationalPublication No. WO 92/16553 (published Oct. 1, 1992)); Knight, D. M. etal. (Mol. Immunol. 30:1443-1453 (1993)); and Siegel, S. A. et al.(Cytokine 7(1):15-25 (1995)). These references are entirely incorporatedherein by reference.

Chimeric A2 anti-TNF consists of the antigen binding variable region ofthe high-affinity neutralizing mouse anti-human TNF IgG1 antibody,designated A2, and the constant regions of a human IgG1, kappaimmunoglobulin. The human IgG1 Fc region improves allogeneic antibodyeffector function, increases the circulating serum half-life anddecreases the immunogenicity of the antibody. The avidity and epitopespecificity of the chimeric A2 is derived from the variable region ofthe murine A2. Chimeric A2 neutralizes the cytotoxic effect of bothnatural and recombinant human TNF in a dose dependent manner. Frombinding assays of cA2 and recombinant human TNF, the affinity constantof cA2 was calculated to be 1.8×10⁹M⁻¹. Preferred methods fordetermining mAb specificity and affinity by competitive inhibition canbe found in Harlow, et al., Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1988; Colligan etal., eds., Current Protocols in Immunology, Greene Publishing Assoc. andWiley Interscience, New York, (1992, 1993); Kozbor et al., Immunol.Today 4:72-79 (1983); Ausubel et al., eds. Current Protocols inMolecular Biology, Wiley Interscience, New York (1987, 1992, 1993); andMuller, Meth. Enzymol. 92:589-601 (1983), which references are entirelyincorporated herein by reference.

As used herein, the term “antigen binding region” refers to that portionof an antibody molecule which contains the amino acid residues thatinteract with an antigen and confer on the antibody its specificity andaffinity for the antigen. The antibody region includes the “framework”amino acid residues necessary to maintain the proper conformation of theantigen-binding residues. Generally, the antigen binding region will beof murine origin. In other embodiments, the antigen binding region canbe derived from other animal species, such as sheep, rabbit, rat orhamster. Preferred sources for the DNA encoding such a non-humanantibody include cell lines which produce antibody, preferably hybridcell lines commonly known as hybridomas. In one embodiment, a preferredhybridoma is the A2 hybridoma cell line.

An “antigen” is a molecule or a portion of a molecule capable of beingbound by an antibody which is additionally capable of inducing an animalto produce antibody capable of selectively binding to an epitope of thatantigen. An antigen can have one or more than one epitope.

The term “epitope” is meant to refer to that portion of the antigencapable of being recognized by and bound by an antibody at one or moreof the antibody's antigen binding region. Epitopes usually consist ofchemically active surface groupings of molecules such as amino acids orsugar side chains and have specific three dimensional structuralcharacteristics as well as specific charge characteristics. By“inhibiting and/or neutralizing epitope” is intended an epitope, which,when bound by an antibody, results in loss of biological activity of themolecule containing the epitope, in vivo or in vitro, more preferably invivo, including binding of TNF to a TNF receptor. Epitopes of TNF havebeen identified within amino acids 1 to about 20, about 56 to about 77,about 108 to about 127 and about 138 to about 149. Preferably, theantibody binds to an epitope comprising at least about 5 amino acids ofTNF within TNF residues from about 87 to about 107, about 59 to about 80or a combination thereof. Generally, epitopes include at least about 5amino acids and less than about 22 amino acids embracing or overlappingone or more of these regions.

For example, epitopes of TNF which are recognized by, and/or binds withanti-TNF activity, an antibody, and fragments, and valuable regionsthereof, include: 59-80: Tyr-Ser-Gln-Val-Leu-Phe-Lys-Gly-Gln-Gly- (SEQID NO:1) Cys-Pro-Ser-Thr-His-Val-Leu-Leu-Thr-His- Thr-Ile; and/or87-108: Tyr-Gln-Thr-Lys-Val-Asn-Leu-Leu-Ser-Ala- (SEQ ID NO:2)Ile-Lys-Ser-Pro-Cys-Gln-Arg-Glu-Thr-Pro- Glu-Gly.

The anti-TNF antibodies, and fragments, and variable regions thereof,that are recognized by, and/or binds with anti-TNF activity, theseepitopes block the action of TNFα without binding to the putativereceptor binding locus as presented by Eck and Sprang (J. Biol. Chem.264(29): 17595-17605 (1989) (amino acids 11-13, 37-42, 49-57 and 155-157of hTNFα). Rathjen et al., International Publication No. WO 91/02078(published Feb. 21, 1991), incorporated herein by reference, disclosesTNF ligands which can bind additional epitopes of TNF.

Antibody Production Using Hybridomas

The techniques to raise antibodies to small peptide sequences thatrecognize and bind to those sequences in the free or conjugated form orwhen presented as a native sequence in the context of a large proteinare well known in the art. Such antibodies can be produced by hybridomaor recombinant techniques known in the art.

Murine antibodies which can be used in the preparation of the antibodiesuseful in the methods and compositions of the present invention havealso been described in Rubin et al., EP 0218868 (published Apr. 22,1987); Yone et al., EP 0288088 (published Oct. 26, 1988); Liang, et al.,Biochem. Biophys. Res. Comm. 137:847-854 (1986); Meager, et al.,Hybridoma 6:305-311 (1987); Fendly et al., Hybridoma 6:359-369 (1987);Bringman, et al., Hybridoma 6:489-507 (1987); Hirai, et al., J. Immunol.Meth. 96:57-62 (1987); Möller, et al., Cytokine 2:162-169 (1990).

The cell fusions are accomplished by standard procedures well known tothose skilled in the field of immunology. Fusion partner cell lines andmethods for fusing and selecting hybridomas and screening for mAbs arewell known in the art. See, e.g, Ausubel infra, Harlow infra, andColligan infra, the contents of which references are incorporatedentirely herein by reference.

The TNFα-specific murine mAb useful in the methods and compositions ofthe present invention can be produced in large quantities by injectinghybridoma or transfectoma cells secreting the antibody into theperitoneal cavity of mice and, after appropriate time, harvesting theascites fluid which contains a high titer of the mAb, and isolating themAb therefrom. For such in vivo production of the mAb with a hybridoma(e.g., rat or human), hybridoma cells are preferably grown in irradiatedor athymic nude mice. Alternatively, the antibodies can be produced byculturing hybridoma or transfectoma cells in vitro and isolatingsecreted mAb from the cell culture medium or recombinantly, ineukaryotic or prokaryotic cells.

In one embodiment, the antibody used in the methods and compositions ofthe present invention is a mAb which binds amino acids of an epitope ofTNF recognized by A2, rA2 or cA2, produced by a hybridoma or by arecombinant host. In another embodiment, the antibody is a chimericantibody which recognizes an epitope recognized by A2. In still anotherembodiment, the antibody is a chimeric antibody designated as chimericA2 (cA2).

As examples of antibodies useful in the methods and compositions of thepresent invention, murine mAb A2 is produced by a cell line designatedc134A.

“Derivatives” of the antibodies including fragments, regions or proteinsencoded by truncated or modified genes to yield molecular speciesfunctionally resembling the immunoglobulin fragments are also useful inthe methods and compositions of the present invention. The modificationsinclude, but are not limited to, addition of genetic sequences codingfor cytotoxic proteins such as plant and bacterial toxins. The fragmentsand derivatives can be produced from appropriate cells, as is known inthe art. Alternatively, anti-TNF antibodies, fragments and regions canbe bound to cytotoxic proteins or compounds in vitro, to providecytotoxic anti-TNF antibodies which would selectively kill cells havingTNF on their surface.

“Fragments” of the antibodies include, for example, Fab, Fab′, F(ab′)₂and Fv. These fragments lack the Fc fragment of intact antibody, clearmore rapidly from the circulation, and can have less non-specific tissuebinding than an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325(1983)). These fragments are produced from intact antibodies usingmethods well known in the art, for example by proteolytic cleavage withenzymes such as papain (to produce Fab fragments) or pepsin (to produceF(ab′)₂ fragments).

Recombinant Expression of Anti-TNF Antibodies

Recombinant and/or chimeric murine-human or human-human antibodies thatinhibit TNF can be produced using known techniques based on theteachings provided in U.S. application Ser. No. 08/192,093 (filed Feb.4, 1994), U.S. application Ser. No. 08/192,102 (filed Feb. 4, 1994; nowU.S. Pat. No. 5,656,272), U.S. application Ser. No. 08/192,861 (filedFeb. 4, 1994; now U.S. Pat. No. 5,919,452), U.S. application Ser. No.08/324,799 (filed on Oct. 18, 1994; now U.S. Pat. No. 5,698,195) and Le,J. et al., International Publication No. WO 92/16553 (published Oct. 1,1992), which references are entirely incorporated her-ein by reference.See, e.g., Ausubel et al., eds. Current Protocols in Molecular Biology,Wiley Interscience, New York (1987, 1992, 1993); and Sambrook et al.Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, New York (1989), the contents of which are entirely incorporatedherein by reference. See also, e.g., Knight, D. M., et al., Mol. Immunol30:1443-1453 (1993); and Siegel, S. A., et al., Cytokine 7(1):15-25(1995), the contents of which are entirely incorporated herein byreference.

The DNA encoding an anti-TNF antibody can be genomic DNA or cDNA whichencodes at least one of the heavy chain constant region (Hc), the heavychain variable region (Hc), the light chain variable region (Lv) and thelight chain constant regions (Lc). A convenient alternative to the useof chromosomal gene fragments as the source of DNA encoding the murine Vregion antigen-binding segment is the use of cDNA for the constructionof chimeric immunoglobulin genes, e.g., as reported by Liu et al. (Proc.Natl. Acad. Sci., USA 84:3439 (1987) and J. Immunology 139:3521 (1987)),which references are entirely incorporated herein by reference. The useof cDNA requires that gene expression elements appropriate for the hostcell be combined with the gene in order to achieve synthesis of thedesired protein. The use of cDNA sequences is advantageous over genomicsequences (which contain introns), in that cDNA sequences can beexpressed in bacteria or other hosts which lack appropriate RNA splicingsystems. An example of such a preparation is set forth below.

Because the genetic code is degenerate, more than one codon can be usedto encode a particular amino acid. Using the genetic code, one or moredifferent oligonucleotides can be identified, each of which would becapable of encoding the amino acid. The probability that a particularoligonucleotide will, in fact, constitute the actual XXX-encodingsequence can be estimated by considering abnormal base pairingrelationships and the frequency with which a particular codon isactually used (to encode a particular amino acid) in eukaryotic orprokaryotic cells expressing an anti-TNF antibody or fragment. Such“codon usage rules” are disclosed by Lathe, et al., J. Mol. Biol.183:1-12 (1985). Using the “codon usage rules” of Lathe, a singleoligonucleotide, or a set of oligonucleotides, that contains atheoretical “most probable” nucleotide sequence capable of encodinganti-TNF variable or constant region sequences is identified.

Although occasionally an amino acid sequence can be encoded by only asingle oligonucleotide, frequently the amino acid sequence can beencoded by any of a set of similar oligonucleotides. Importantly,whereas all of the members of this set contain oligonucleotides whichare capable of encoding the peptide fragment and, thus, potentiallycontain the same oligonucleotide sequence as the gene which encodes thepeptide fragment, only one member of the set contains the nucleotidesequence that is identical to the nucleotide sequence of the gene.Because this member is present within the set, and is capable ofhybridizing to DNA even in the presence of the other members of the set,it is possible to employ the unfractionated set of oligonucleotides inthe same manner in which one would employ a single oligonucleotide toclone the gene that encodes the protein.

The oligonucleotide, or set of oligonucleotides, containing thetheoretical “most probable” sequence capable of encoding an anti-TNFantibody or fragment including a variable or constant region is used toidentify the sequence of a complementary oligonucleotide or set ofoligonucleotides which is capable of hybridizing to the “most probable”sequence, or set of sequences. An oligonucleotide containing such acomplementary sequence can be employed as a probe to identify andisolate the variable or constant region anti-TNF gene (Sambrook et al.,infra).

A suitable oligonucleotide, or set of oligonucleotides, which is capableof encoding a fragment of the variable or constant anti-TNF region (orwhich is complementary to such an oligonucleotide, or set ofoligonucleotides) is identified (using the above-described procedure),synthesized, and hybridized by means well known in the art, against aDNA or, more preferably, a cDNA preparation derived from cells which arecapable of expressing anti-TNF antibodies or variable or constantregions thereof. Single stranded oligonucleotide molecules complementaryto the “most probable” variable or constant anti-TNF region peptidecoding sequences can be synthesized using procedures which are wellknown to those of ordinary skill in the art (Belagaje, et al., J. Biol.Chem. 254:5765-5780 (1979); Maniatis, et al., In: Molecular Mechanismsin the Control of Gene Expression, Nierlich, et al., eds., Acad. Press,New York (1976); Wu, et al., Prog. Nucl. Acid Res. Molec. Biol.21:101-141 (1978); Khorana, Science 203:614-625 (1979)). Additionally,DNA synthesis can be achieved through the use of automated synthesizers.Techniques of nucleic acid hybridization are disclosed by Sambrook etal., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, New York (1989); and by Haynes, et al., in: NucleicAcid Hybridization, A Practical Approach, IRL Press, Washington, D.C.(1985), which references are entirely incorporated herein by reference.Techniques such as, or similar to, those described above havesuccessfully enabled the cloning of genes for human aldehydedehydrogenases (Hsu, et al., Proc. Natl. Acad. Sci. USA 82:3771-3775(1985)), fibronectin (Suzuki, et al., Bur. Mol. Biol. Organ. J.4:2519-2524 (1985)), the human estrogen receptor gene (Walter, et al.,Proc. Natl. Acad. Sci. USA 82:7889-7893 (1985)), tissue-type plasminogenactivator (Pennica, et al., Nature 301:214-221 (1983)) and humanplacental alkaline phosphatase complementary DNA (Keun, et al., Proc.Natl. Acad. Sci. USA 82:8715-8719 (1985)).

In an alternative way of cloning a polynucleotide encoding an anti-TNFvariable or constant region, a library of expression vectors is preparedby cloning DNA or, more preferably, cDNA (from a cell capable ofexpressing an anti-TNF antibody or variable or constant region) into anexpression vector. The library is then screened for members capable ofexpressing a protein which competitively inhibits the binding of ananti-TNF antibody, such as A2 or cA2, and which has a nucleotidesequence that is capable of encoding polypeptides that have the sameamino acid sequence as anti-TNF antibodies or fragments thereof. In thisembodiment, DNA, or more preferably cDNA, is extracted and purified froma cell which is capable of expressing an anti-TNF antibody or fragment.The purified cDNA is fragmentized (by shearing, endonuclease digestion,etc.) to produce a pool of DNA or cDNA fragments. DNA or cDNA fragmentsfrom this pool are then cloned into an expression vector in order toproduce a genomic library of expression vectors whose members eachcontain a unique cloned DNA or cDNA fragment such as in a lambda phagelibrary, expression in prokaryotic cell (e.g., bacteria) or eukaryoticcells, (e.g., mammalian, yeast, insect or, fungus). See, e.g., Ausubel,infra, Harlow, infra, Colligan, infra; Nyyssonen et al. Bio/Technology11:591-595 (1993); Marks et al., Bio/Technology 11:1145-1149 (October1993). Once nucleic acid encoding such variable or constant anti-TNFregions is isolated, the nucleic acid can be appropriately expressed ina host cell, along with other constant or variable heavy or light chainencoding nucleic acid, in order to provide recombinant monoclonalantibodies that bind TNF with inhibitory activity. Such antibodiespreferably include a murine or human anti-TNF variable region whichcontains a framework residue having complementarity determining residueswhich are responsible for antigen binding.

Human genes which encode the constant (C) regions of the chimericantibodies, fragments and regions of the present invention can bederived from a human fetal liver library, by known methods. Human Cregion genes can be derived from any human cell including those whichexpress and produce human immunoglobulins. The human CH region can bederived from any of the known classes or isotypes of human H chains,including gamma, μ, α, δ or ε, and subtypes thereof, such as G1, G2, G3and G4. Since the H chain isotype is responsible for the variouseffector functions of an antibody, the choice of CH region will beguided by the desired effector functions, such as complement fixation,or activity in antibody-dependent cellular cytotoxicity (ADCC).Preferably, the CH region is derived from gamma 1 (IgG1), gamma 3(IgG3), gamma 4 (IgG4), or μ (IgM). The human CL region can be derivedfrom either human L chain isotype, kappa or lambda.

Genes encoding human immunioglobulin C regions are obtained from humancells by standard cloning techniques (Sambrook, et al. (MolecularCloning. A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press,Cold Spring Harbor, N.Y. (1989) and Ausubel et al., eds., CurrentProtocols in Molecular Biology, Wiley Interscience, New York(1987-1993)). Human C region genes are readily available from knownclones containing genes representing the two classes of L chains, thefive classes of H chains and subclasses thereof. Chimeric antibodyfragments, such as F(ab′)₂ and Fab, can be prepared by designing achimeric H chain gene which is appropriately truncated. For example, achimeric gene encoding an H chain portion of an F(ab′)₂ fragment wouldinclude DNA sequences encoding the CH1 domain and hinge region of the Hchain, followed by a translational stop codon to yield the truncatedmolecule.

Generally, the murine, human and chimeric antibodies, fragments andregions are produced by cloning DNA segments encoding the H and L chainantigen-binding regions of a TNF-specific antibody, and joining theseDNA segments to DNA segments encoding CH and CL regions, respectively,to produce murine, human or chimeric immunoglobulin-encoding genes.Thus, in a preferred embodiment, a fused chimeric gene is created whichcomprises a first DNA segment that encodes at least the antigen-bindingregion of non-human origin, such as a functionally rearranged V regionwith joining (J) segment, linked to a second DNA segment encoding atleast a pall of a human C region.

Therefore, cDNA encoding the antibody J and C regions and the method ofproducing a chimeric antibody can involve several steps, outlined below:

-   -   1. Isolation of messenger RNA (mRNA) from the cell line        producing an anti-TNF antibody and from optional additional        antibodies supplying heavy and light constant regions; cloning        and cDNA production therefrom;    -   2. Preparation of a full length cDNA library from purified mRNA        from which the appropriate V and/or C region gene segments of        the L and H chain genes can be: (i) identified with appropriate        probes, (ii) sequenced, and (iii) made compatible with a C or V        gene segment from another antibody for a chimeric antibody;    -   3. Construction of complete H or L chain coding sequences by        linkage of the cloned specific V region gene segments to cloned        C region gene, as described above;    -   4. Expression and production of L and H chains in selected        hosts, including prokaryotic and eukaryotic cells to provide        murine-murine, human-murine, human-human or human-murine        antibodies.

One common feature of all immunoglobulin H and L chain genes and theirencoded mRNTAs is the J region. H and L chain J regions have differentsequences, but a high degree of sequence homology exists (greater than80%) among each group, especially near the C region. This homology isexploited in this method and consensus sequences of H and L chain Jregions can be used to design oligonucleotides for use as primers forintroducing useful restriction sites into the J region for subsequentlinkage of V region segments to human C region segments.

C region cDNA vectors prepared from human cells can be modified bysite-directed mutagenesis to place a restriction site at the analogousposition in the human sequence. For example, one can clone the completehuman kappa chain C (Ck) region and the complete human gamma-1 C region(C gamma-1). In this case, the alternative method based upon genomic Cregion clones as the source for C region vectors would not allow thesegenes to be expressed in bacterial systems where enzymes needed toremove intervening sequences are absent. Cloned V region segments areexcised and ligated to L or H chain C region vectors. Alternatively, thehuman C gamma-1 region can be modified by introducing a terminationcodon thereby generating a gene sequence which encodes the H chainportion of an Fab molecule. The coding sequences with linked V and Cregions are then transferred into appropriate expression vehicles forexpression in appropriate hosts, prokaryotic or eukaryotic.

Two coding DNA sequences are said to be “operably linked” if the linkageresults in a continuously translatable sequence without alteration orinterruption of the triplet reading frame. A DNA coding sequence isoperably linked to a gene expression element if the linkage results inthe proper function of that gene expression element to result inexpression of the coding sequence.

Expression vehicles include plasmids or other vectors. Preferred amongthese are vehicles carrying a functionally complete human CH or CL chainsequence having appropriate restriction sites engineered so that any VHor VL chain sequence with appropriate cohesive ends can be easilyinserted therein. Human CH or CL chain sequence-containing vehicles thusserve as intermediates for the expression of any desired complete H or Lchain in any appropriate host.

A chimeric antibody, such as a mouse-human or human-human, willtypically be synthesized from genes driven by the chromosomal genepromoters native to the mouse H and L chain V regions used in theconstructs; splicing usually occurs between the splice donor site in themouse J region and the splice acceptor site preceding the human C regionand also at the splice regions that occur within the human C, region;polyadenylation and transcription termination occur at nativechromosomal sites downstream of the human coding regions.

A nucleic acid sequence encoding at least one anti-TNF antibody fragmentmay be recombined with vector DNA in accordance with conventionaltechniques, including blunt-ended or staggered-ended termini forligation, restriction enzyme digestion to provide appropriate termini,filling in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining, and ligation with appropriateligases. Techniques for such manipulations are disclosed, e.g., byAusubel, supra, Sambrook, supra, entirely incorporated herein byreference, and are well known in the art.

A nucleic acid molecule, such as DNA, is “capable of expressing” apolypeptide if it contains nucleotide sequences which containtranscriptional and translational regulatory information and suchsequences are “operably linked” to nucleotide sequences which encode thepolypeptide. An operable linkage is a linkage in which the regulatoryDNA sequences and the DNA sequence sought to be expressed are connectedin such a way as to permit gene expression as anti-TNF peptides orantibody fragments in recoverable amounts. The precise nature of theregulatory regions needed for gene expression may vary from organism toorganism and is well known in the analogous art. See, e.g., Sambrook etal., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, New York (1989); and Ausubel, eds., Current Protocolsin Molecular Biology, Wiley Interscience, New York (1987, 1993).

Many vector systems are available for the expression of cloned anti-TNFpeptide H and L chain genes in mammalian cells (see Glover, ed., DNACloning, Vol. II, pp. 143-238, IRL Press, Washington, D.C., 1985).Different approaches can be followed to obtain complete H2L2 antibodies.It is possible to co-express H and L chains in the same cells to achieveintracellular association and linkage of H and L chains into completetetrameric H2L2 antibodies. The co-expression can occur by using eitherthe same or different plasmids in the same host. Genes for both H and Lchains can be placed into the same plasmid, which is then transfectedinto cells, thereby selecting directly for cells that express bothchains. Alternatively, cells can be transfected first with a plasmidencoding one chain, for example the L chain, followed by transfection ofthe resulting cell line with an H chain plasmid containing a secondselectable marker. Cell lines producing H2L2 molecules via either routecould be transfected with plasmids encoding additional copies ofpeptides, H, L, or H plus L chains in conjunction with additionalselectable markers to generate cell lines with enhanced properties, suchas higher production of assembled H2L2 antibody molecules or enhancedstability of the transfected cell lines.

Receptor Molecules

Receptor molecules (also referred to herein as soluble TNF receptors)useful in the methods and compositions of the present invention arethose that bind TNF with high affinity (see, e.g., Feldman et al.,International Publication No. WO 92/07076 (published Apr. 30, 1992),incorporated herein by reference) and possess low immunogenicity. Inparticular, the 55 kDa (p55 TNF-R) and the 75 kDa (p75 TNF-R) TNF cellsurface receptors are useful in the present invention. Truncated formsof these receptors, comprising the extracellular domains (ECD) of thereceptors or functional portions thereof, are also useful in the presentinvention. Truncated forms of the TNF receptors, comprising the ECD,have been detected in urine and serum as 30 kDa and 40 kDa TNFinhibitory binding proteins (Engelmann, H. et al., J. Biol. Chem.265:1531-1536 (1990)). TNF receptor multimeric molecules and TNFimmunoreceptor fusion molecules, and derivatives and fragments orportions thereof, are additional examples of receptor molecules whichare useful in the methods and compositions of the present invention. Thereceptor molecules which can be used in the invention are characterizedby their ability to treat patients for extended periods with good toexcellent alleviation of symptoms and low toxicity. Low immunogenicityand/or high affinity, as well as other undefined properties, maycontribute to the therapeutic results achieved.

TNF receptor multimeric molecules useful in the present inventioncomprise all or a functional portion of the ECD of two or more TNFreceptors linked via one or more polypeptide linkers. The multimericmolecules can further comprise a signal peptide of a secreted protein todirect expression of the multimeric molecule. These multimeric moleculesand methods for their production have been described in U.S. applicationSer. No. 08/437,533 (filed May 9, 1995), the content of which isentirely incorporated herein by reference.

TNF immunoreceptor fusion molecules useful in the methods andcompositions of the present invention comprise at least one portion ofone or more immunoglobulin molecules and all or a functional portion ofone or more TNF receptors. These immunoreceptor fusion molecules can beassembled as monomers, or hetero- or homo-multimers. The immunoreceptorfusion molecules can also be monovalent or multivalent. An example ofsuch a TNF immunoreceptor fusion molecule is TNF receptor/IgG fusionprotein.

TNF immunoreceptor fusion molecules and methods for their productionhave been described in the art (Lesslauer et al., Eur. J. Immunol.21:2883-2886 (1991); Ashkenazi et al., Proc. Natl. Acad. Sci. USA88:10535-10539 (1991); Peppel et al., J. Exp. Med. 174:1483-1489 (1991);Kolls et al., Proc. Natl. Acad. Sci. USA 91:215-219 (1994); Butler etal., Cytokine 6(6):616-623 (1994); Baker et al., Eur. J. Immunol.24:2040-2048 (1994); Beutler et al., U.S. Pat. No. 5,447,851; and U.S.application Ser. No. 08/442,133 (filed May 16, 1995)). These referencesare entirely incorporated herein by reference. Methods for producingimmunoreceptor fusion molecules can also be found in Capon et al., U.S.Pat. No. 5,116,964; Capon et al., U.S. Pat. No. 5,225,538; and Capon etal., Nature 337:525-531 (1989), which references are entirelyincorporated herein by reference.

Derivatives, fragments, regions and functional portions of the receptormolecules functionally resemble the receptor molecules that can be usedin the present invention (i.e., they bind TNF with high affinity andpossess low immunogenicity). A functional equivalent or derivative ofthe receptor molecule refers to the portion of the receptor molecule, orthe portion of the receptor molecule sequence which encodes the receptormolecule, that is of sufficient size and sequences to functionallyresemble the receptor molecules that can be used in the presentinvention (i.e., bind TNF with high affinity and possess lowimmunogenicity). A functional equivalent of the receptor molecule alsoincludes modified receptor molecules that functionally resemble thereceptor molecules that can be used in the present invention (i.e., bindTNF with high affinity and possess low immunogenicity). For example, afunctional equivalent of the receptor molecule can contain a “SILENT”codon or one or more amino acid substitutions, deletions or additions(e.g., substitution of one acidic amino acid for another acidic aminoacid; or substitution of one codon encoding the same or differenthydrophobic amino acid for another codon encoding a hydrophobic aminoacid). See Ausubel et al., Current Protocols in Molecular Biology,Greene Publishing Assoc. and Wiley-Interscience, New York (1989).

Methotrexate

Presently available oral and intravenous formulations of methotrexateinclude RHEUMATREX® methotrexate dose pack (Lederle Laboratories, Wayne,N.J.); methotrexate tablets (Mylan Pharmaceuticals Inc., Morgantown, W.Va.; Roxane Laboratories, Inc., Columbus, Ohio); and methotrexate sodiumtablets, for injection and injection (Immunex Corporation, Seattle,Wash.) and methotrexate LPF® sodium (methotrexate sodium injection)(Immunex Corporation, Seattle, Wash.). Methotrexate is also availablefrom Pharmacochemie (Netherlands). Methotrexate prodrugs, homologsand/or analogs (e.g., folate antagonists) can also be used in themethods and compositions of the present invention. Alternatively, otherimmunosuppressive agents (or drugs that suppress the immune system) canbe used in the methods and compositions of the present invention.

Administration

TNF antagonists, methotrexate and the compositions of the presentinvention can be administered to an individual in a variety of ways. Theroutes of administration include intradermal, transdermal (e.g., in slowrelease polymers), intramuscular, intraperitoneal, intravenous,subcutaneous, oral, topical, epidural, buccal, rectal, vaginal andintranasal routes. Any other therapeutically efficacious route ofadministration can be used, for example, infusion or bolus injection,absorption through epithelial or mucocutaneous linings, or by genetherapy wherein a DNA molecule encoding the therapeutic protein orpeptide is administered to the patient, e.g., via a vector, which causesthe protein or peptide to be expressed and secreted at therapeuticlevels in vivo. In addition, the TNF antagonists, methotrexate andcompositions of the present invention can be administered together withother components of biologically active agents, such as pharmaceuticallyacceptable surfactants (e.g., glycerides), excipients (e.g., lactose),carriers, diluents and vehicles. If desired, certain sweetening,flavoring and/or coloring agents can also be added.

The TNF antagonists and methotrexate can be administeredprophylactically or therapeutically to an individual. TNF antagonistscan be administered prior to, simultaneously with (in the same ordifferent compositions) or sequentially with the administration ofmethotrexate. For example, TNF antagonists can be administered asadjunctive and/or concomitant therapy to methotrexate therapy.

For parenteral (e.g., intravenous, subcutaneous, intramuscular)administration, TNF antagonists, methotrexate and the compositions ofthe present invention can be formulated as a solution, suspension,emulsion or lyophilized powder in association with a pharmaceuticallyacceptable parenteral vehicle. Examples of such vehicles are water,saline, Ringer's solution, dextrose solution, and 5% human serumalbumin. Liposomes and nonaqueous vehicles such as fixed oils can alsobe used. The vehicle or lyophilized powder can contain additives thatmaintain isotonicity (e.g., sodium chloride, mannitol) and chemicalstability (e.g., buffers and preservatives). The formulation issterilized by commonly used techniques.

Suitable pharmaceutical carriers are described in Remington'sPharmaceutical Sciences, A. Osol, a standard reference text in thisfield of art.

For example, a parenteral composition suitable for administration byinjection is prepared by dissolving 1.5% by weight of active ingredientin 0.9% sodium chloride solution.

TNF antagonists and methotrexate are administered in therapeuticallyeffective amounts; the compositions of the present invention areadministered in a therapeutically effective amount. As used herein, a“therapeutically effective amount” is such that administration of TNFantagonist and methotrexate, or administration of a composition of thepresent invention, results in inhibition of the biological activity ofTNF relative to the biological activity of TNF when therapeuticallyeffective amounts of antagonist and methotrexate are not administered,or relative to the biological activity of TNF when a therapeuticallyeffective amount of the composition is not administered. Atherapeutically effective amount is preferably an amount of TNFantagonist and methotrexate necessary to significantly reduce oreliminate signs and symptoms associated with a particular TNF-mediateddisease. As used herein, a therapeutically effective amount is notnecessarily an amount such that administration of the TNF antagonistalone, or administration of methotrexate alone, must necessarily resultin inhibition of the biological activity of TNF.

Once a therapeutically effective amount has been administered, amaintenance amount of TNF antagonist alone, of methotrexate alone, or ofa combination of TNF antagonist and methotrexate can be administered tothe individual. A maintenance amount is the amount of TNF antagonist,methotrexate, or combination of TNF antagonist and methotrexatenecessary to maintain the reduction or elimination of the signs andsymptoms associated with a particular TNF-mediated disease achieved bythe therapeutically effective dose. The maintenance amount can beadministered in the form of a single dose, or a series or dosesseparated by intervals of days or weeks.

The dosage administered to an individual will vary depending upon avariety of factors, including the pharmacodynamic characteristics of theparticular antagonists, and its mode and route of administration; size,age, sex, health, body weight and diet of the recipient; nature andextent of symptoms of the disease being treated, kind of concurrenttreatment, frequency of treatment, and the effect desired. In vitro andin vivo methods of determining the inhibition of TNF in an individualare well known to those of skill in the art. Such in vitro assays caninclude a TNF cytotoxicity assay (e.g., the WEHI assay or aradioimmunoassay, ELISA). In vivo methods can include rodent lethalityassays and/or primate pathology model systems (Mathison et al., J. Clin.Invest., 81:1925-1937 (1988); Beutler et al., Science 229:869-871(1985); Tracey et al., Nature 330:662-664 (1987); Shimamoto et al.,Immunol. Lett. 17:311-318 (1988); Silva et al., J. Infect. Dis.162:421-427 (1990); Opal et al., J. Infect. Dis. 161:1148-1152 (1990);Hinshaw et al., Circ. Shock 30:279-292 (1990)).

TNF antagonist and methotrexate can each be administered in single ormultiple doses depending upon factors such as nature and extent ofsymptoms, kind of concurrent treatment and the effect desired. Thus,other therapeutic regimens or agents (e.g., multiple drug regimens) canbe used in combination with the therapeutic co-administration of TNFantagonists and methotrexate. In a particular embodiment, a TNFantagonist is administered in multiple doses. In another embodiment,methotrexate is administered in the form of a series of low dosesseparated by intervals of days or weeks. Adjustment and manipulation ofestablished dosage ranges are well within the ability of those skilledin the art.

Usually a daily dosage of active ingredient can be about 0.01 to 100milligrams per kilogram of body weight. Ordinarily 1 to 40 milligramsper kilogram per day given in divided doses 1 to 6 times a day or insustained release form is effective to obtain desired results. Second orsubsequent administrations can be administered at a dosage which is thesame, less than or greater than the initial or previous doseadministered to the individual.

A second or subsequent administration is preferably during orimmediately prior to relapse or a flare-up of the disease or symptoms ofthe disease. For example, second and subsequent administrations can begiven between about one day to 30 weeks from the previousadministration. Two, three, four or more total administrations can bedelivered to the individual, as needed.

Dosage forms (composition) suitable for internal administrationgenerally contain from about 0.1 milligram to about 500 milligrams ofactive ingredient per unit. In these pharmaceutical compositions theactive ingredient will ordinarily be present in an amount of about0.5-95% by weight based on the total weight of the composition.

The present invention will now be illustrated by the following example,which is not intended to be limiting in any way.

EXAMPLES Example 1 Clinical Treatment of Rheumatoid Arthritis byMultiple Infusions of an Anti-TNF Antibody with and without Methotrexate

A randomized, double-blind, placebo controlled study was conducted toevaluate the safety and efficacy of a chimeric monoclonal anti-TNFantibody (cA2) following multiple infusions of 1, 3 or 10 mg/kg cA2,alone or in combination with methotrexate, compared to multipleinfusions of placebo in combination with methotrexate, in the treatmentof rheumatoid arthritis (RA) in patients.

Patients

One hundred one (101) patients at six European centers who had beenusing methotrexate for at least 6 months, had been on a stable dose of7.5 mg/wk for at least 4 weeks, and had active disease (according to thecriteria of the American College of Rheumatology) with erosive changeson X-rays of hands and feet, were enrolled in the trial. Active diseasewas defined by the presence of six or more swollen joints plus at leastthree of four secondary criteria (duration of morning stiffness ≧45minutes; ≧6 tender or painful joints; erythrocyte sedimentation rate(ESR) ≧28 mm/hour; C-reactive protein (CRP) ≧20 mg/l.

In patients using corticosteroids (≦7.5 mg/day) or non-steroidalanti-inflammatory drugs (NSAIDs), the doses had been stable for 4 weeksprior to screening. The dose of corticosteroids remained stablethroughout trial participation. The dose of NSAID typically alsoremained stable throughout trial participation.

Study Infusions

The chimeric monoclonal anti-TNF antibody (cA2) was supplied as asterile solution containing 5 mg cA2 per ml of 0.01 M phosphate-bufferedsaline in 0.15 M sodium chloride with 0.01% polysorbate 80, pH 7.2. Theplacebo vials contained 0.1% human serum albumin in the same buffer.Before use, the appropriate amount of cA2 or placebo was diluted to 300ml in sterile saline by the pharmacist, and administered intravenouslyvia a 0.2 μm in-line filter over 2 hours. The characteristics of theplacebo and cA2 infusion bags were identical, and the investigators andpatients did not know which infusion was being administered.

Assessments

Patients were randomized to one of seven treatment groups. The number ofpatients in each dose (or treatment) group is indicated in Table 1. Eachof the 101 patients received multiple infusions of either 0, 1, 3, or1.0 mg/kg cA2. Infusions were to be administered at weeks O, 2, 6, 10and 14. Starting at week 0, the patients were receiving 7.5 mg/wk ofmethotrexate (Pharmacochemie, Netherlands) or 3 placebo tablets/week(Pharmacochemie, Netherlands). Patients were monitored for adverseevents during infusions and regularly thereafter, by interviews,physical examination, and laboratory testing.

The six primary disease-activity assessments were chosen to allowanalysis of the response in individual patients according to the Paulusindex (Paulus, et al., Arthritis Rheumatism 33:477-484 (1990), theteachings of which are incorporated herein by reference). Theassessments contributing to this index were the tender joint and swollenjoint scores (60 and 58 joints, respectively, hips not assessed forswelling; graded 0-3), the duration of morning stiffness (minutes), thepatient's and physician's assessment of disease severity (on a 5-pointscale, ranging from 1 (symptom-free) to 5 (very severe), and erythrocytesedimentation rate (ESR). Patients were considered to have responded ifat least four of the six variables improved, defined as at least 20%improvement in the continuous variables, and at least two grades ofimprovement or improvement from grade 2 to 1 in the two disease-severityassessments (Paulus 20% response). Improvements of at least 50% in thecontinuous variables were also used (Paulus 50% response).

Other disease-activity assessments included the pain score (0-10 cm on avisual analogue scale (VAS)), an assessment of fatigue (0-10 cm VAS),and grip strength (0-300 mm Hg, mean of three measurements per hand bysphygmomanometer cuff).

The ESR was measured at each study site with a standard method(Westergen). C-reactive protein (CRP) was measured by rate nephelometry(Abbott fluorescent polarizing immunoassay). See also, Elliott et al.,Lancet 344:1105-1110 (1994); Elliott et al., Lancet 344:1125-1127(1994); and Elliott et al., Arthritis Rheum. 36(12): 1681-1690 (1993),which references are entirely incorporated herein by reference.

Evaluations were performed at weeks 1, 2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22 and 26.

Results

The 101 patients were randomized to one of seven treatment (or dose)groups. The patients enrolled in each dose group were well matched forbaseline demographics. Disease duration and swollen and tender jointcounts at baseline were also well-balanced across the groups (Table 1).Table 1 also shows the maximum methotrexate dose administered within 6months prior to randomization. Median maximum doses for each groupranged between 10 and 15 mg/week; there were no significant differencesamongst the treatment groups (p=0.404). TABLE 1 Baseline DiseaseCharacteristics Joint Counts Treatment Groups Treatment Placebo 1 mg/kgcA2 3 mg/kg cA2 10 mg/kg cA2 All effect MTX+ MTX+ MTX− MTX+ MTX− MTX+MTX− patients p-value Disease dur. (yrs) Pts evaluated 14 14 15 15 14 1415 101 0.634 Mean ± SD 7.6 ± 4.0 14.3 ± 12.1 7.6 ± 6.0 12.1 ± 9.0 7.8 ±4.3 11.1 ± 7.4 9.7 ± 7.4 10.0 ± 7.8 Median 6.9 11.4 11.9 7.7 10.7 7.67.6 IQ range (4.3, 11.5) (3.3, 24.7) (3.4, 9.0) (4.3, 16.4) (4.6,9.8)(4.5, 15.5) (4.9, 14.9) (4.3, 14.4) Range (1.8, 14.2) (0.7, 37.3) (2.5,21.3) (0.7, 30.5) (1.4, 17.4) (1.4, 24.1) (1.1, 24.3) (0.7, 37.3) Numberof Swollen joints, Paulus joint set (0-58) Pts evaluated 14 14 15 15 1414 15 101 0.643 Mean ± SD 18.1 ± 8.6 16.9 ± 7.8 21.2 ± 11.2 17.7 ± 5.919.7 ± 9.9 21.1 ± 8.2 17.8 ± 8.7 18.9 ± 8.7 Median 16.5 15.5 20.0 16.017.0 19.5 17.0 18.0 IQ range (12.0, 25.0) (10.0, 25.0) (10.0, 33.0)(13.0, 22.0) (11.0, 32.0) (15.0,31.0) (11.0,21.0) (12.0,25.0) Range(6.0, 38.0) (6.0, 29.0) (7.0, 40.0) (10.0, 29.0) (8.0, 34.0) (10.0,34.0) (7.0, 41.0) (6.0, 41.0) Number of tender joints, Paulus joint set(0-60) Pts evaluated 14 14 15 15 14 14 15 101 0.135 Mean ± SD 31.5 ±14.2 19.1 ± 10.7 29.9 ± 17.1 24.5 ± 14.4 31.2 ± 11.7 26.5 ± 12.0 26.2 ±11.7 27.0 ± 13.5 Median 27.0 16.0 30.0 21.0 31.0 25.5 23.0 25.0 IQ range(22.0, 44.0) (13.0, 30.0) (14.0, 45.0) (12.0,32.0) (23.0,39.0) (21.0,38.0) (17.0, 35.0) (15.0, 38.0) Range (8.0, 52.0) (2.0, 39.0) (6.0,58.0) (10.0, 52.0) (9.0, 52.0) (8.0, 44.0) (11.0, 48.0) (2.0, 58.0) Maxdose MTX prev. 6 mo (mg/kg) Pts evaluated 14 14 15 14 13 14 15 99 0.404Mean ± SD 13.8 ± 3.9 11.6 ± 3.5 12.8 ± 5.6 11.6 ± 3.3 11.7 ± 4.8 12.7 ±5.0 12.5 ± 3.0 12.4 ± 4.2 Median 15.0 11.3 12.5 10.0 10.0 10.0 12.5 12.5IQ range (10.0, 15.0) (10.0, 12.5) (10.0, 15.0) (10.0, 15.0) (7.5, 12.5)(10.0, 15.0) (10.0, 15.0) (10.0, 15.0) Range (7.5, 20.0) (7.5, 20.0)(7.5, 30.0) (7.5, 17.5) (7.5, 25.0) (7.5, 25.0) (7.5, 20.0) (7.5, 30.0)MTX = Methotrexate

The pre-specified primary analysis in this trial was the comparison ofthe total time of clinical response during the 26-week follow-up period.The results for the primary analysis are shown in Table 2. The durationof response of all cA2-treated groups, with the exception of the 1 mg/kggroup not receiving methotrexate, was significantly improved (p<0.001)compared to the placebo group receiving methotrexate alone. TABLE 2Total Time of Response^(a) Based On Paulus 20% Criteria Treatment GroupsPlacebo 1 mg/kg cA2 3 mg/kg cA2 10 mg/kg cA2 Treatment Total time ofMTX+ MTX+ MTX− MTX+ MTX− MTX+ MTX− effect response in weeks (n = 14) (n= 14) (n = 15) (n = 15) (n = 14) (n = 13) (n = 15) p-value Median 0 16.62.6 16.5 172 >23.1 10.4 <0.001 Minimum 0 0 0 0 0 0 0 25th percentile 0.06.2 2.0 7.0 4.0 2.6 6.9 75th percentile 0.0 22.5 8.0 >20.120.7 >24.6 >23.1 Maximum >15.1 >26.9 15.1 >24.9 >25.9 >25.6 >26.4p-value vs. MTX alone <0.001 0.119 <0.001 <0.001 <0.001 <0.001^(a)Patients were followed through 26 weeks following the initialinfusion of cA2

The response rates at Paulus 20% are shown in Table 3. Drop-outs wereconsidered as non-responders subsequent to their dropping out from thestudy. With the exception of the 1 mg/kg group not receivingmethotrexate, all of the cA2-treated groups demonstrated clinicalbenefit thorough 14 weeks when the last dose of cA2 was received.Sustained clinical benefit was observed through 26 weeks (the lastfollow-up visit) in patients who received 3 or 10 mg/kg cA2 withmethotrexate. Approximately one-half of the patients who received 3mg/kg cA2 with methotrexate demonstrated continued clinical benefit at26 weeks. TABLE 3 Number of Patients Responding According To Paulus 20%Criteria At Each Evaluation Visit Treatment Groups Placebo mg/kg cA2 3mg/kg cA2 10 mg/kg cA2 Treatment MTX+ MTX+ MTX− MTX+ MTX− MTX+ MTX−effect (n=14) (n=14) (n=15) (n=15) (n=14) (n=13) (n=15) p-value Pts withany response 21% 93% 80% 80% 79% 85% 80% <0.001 (3/14) 13/14 12/15 12/1511/14 11/13 12/15 p-value vs MTX alone <0.001 0.006 0.002 0.002 0.0010.004 Time post-infusion 1 Week 0% 31% 53% 27% 43% 31% 60% (0/14) (4/13)(8/15) (4/15) (6/14) (4/13) (9/15) 2 Weeks 7% 64% 57% 27% 43% 62% 53%(1/14) (9/14) (8/14) (4/15) (6/14) (8/13) (8/15) 4 Weeks^(a) 0% 79% 33%40% 64% 54% 53% 0.002 (0/14) (11/14) (5/15) (6/15) (9/14) (7/13) (8/15)6 Weeks 0% 71% 27% 47% 50% 54% 47% (0/14) (10/14) (4/15) (7/15) (7/14)(7/13) (7/15) 8 Weeks^(a) 14% 64% 20% 60% 71% 69% 40% 0.003 (2/14)(9/14) (3/15) (9/15) (10/14) (9/13) (6/15) 10 Weeks 7% 71% 20% 67% 64%69% 53% (1/14) (10/14) (3/15) (10/15) (9/14) (9/13) (8/15) 12 Weeks^(a)7% 57% 13% 67% 64% 62% 60% <0.001 (1/14) (8/14) (2/15) (10/15) (9/14)(8/13) (8/13) 14 Weeks 0% 71% 7% 60% 57% 77% 53% (0/14) (10/14) (1/15)(9/15) (8/14) 10/13 (8/15) 16 Weeks^(a) 14% 64% 7% 67% 64% 54% 67%<0.001 (2/14) (9/14) (1/15) (10/15) (9/14) (7/13) (10/15) 18 Weeks 21%50% 13% 71% 69% 62% 57% (3/14) (7/14) (2/15) (10/14) (9/13) (8/13)(8/14) 20 Weeks 7% 54% 13% 53% 43% 54% 53% (1/14) (7/13) (2/15) (8/15)(6/14) (7/13) (8/15) 22 Weeks 7% 46% 0% 47% 36% 54% 33% (1/14) (6/13)(0/15) (7/15) (5/14) (7/13) (5/15) 26 Weeks^(a) 7% 21% 7% 47% 21% 54%33% 0.013 (1/14) (3/14) (1/15) (7/15) (3/14) (7/13) (5/15)^(a)Evaluation visits pre-specified for analysis. The response rates atPaulus 50% are shown in Table 4. The magnitude of the clinical benefitof cA2 treatment was substantial. The majority of patients wereresponding to cA2 treatment according to the 50% Paulus criteria.

TABLE 4 Number of Patients Responding According To Paulus 50% CriteriaAt Each Evaluation Visit Treatment Groups Placebo mg/kg cA2 3 mg/kg cA210 mg/kg cA2 Treatment MTX+ MTX+ MTX− MTX+ MTX− MTX+ MTX− effect (n=14)(n=14) (n=15) (n=15) (n=14) (n=13) (n=15) p-value Pts with any response14.3% 85.7% 40.0% 73.3% 64.3% 76.9% 66.7% <0.001 (2/14) 12/14 6/15 11/159/14 10/13 10/15 p-value vs MTX alone <0.001 0.079 0.002 0.002 0.0010.004 Time post-infusion 1 Week 0.0% 7.7% 26.7% 0.0% 35.7% 7.7% 26.7%(0/14) (1/13) (4/15) (0/15) (5/14) (1/13) (4/15) 2 Weeks 0.0% 21.4%28.6% 6.7% 28.6% 15.4% 20.0% (0/14) (3/14) (4/14) (1/15) (4/14) (2/13)(3/15) 4 Weeks^(a) 0.0% 57.1% 13.3% 13.3% 28.6% 46.2% 40.0% 0.006 (0/14)(8/14) (2/15) (2/15) (4/14) (6/13) (6/15) 6 Weeks 0.0% 57.1% 0.0% 26.7%42.9% 38.5% 33.3% (0/14) (8/14) (0/15) (4/15) (6/14) (5/13) (5/15) 8Weeks^(a) 7.1% 50.0% 0.0% 40.0% 50.0% 69.2% 33.3% 0.001 (1/14) (7/14)(0/15) (6/15) (7/14) (9/13) (5/15) 10 Weeks 0.0% 57.1% 0.0% 40.0% 50.0%69.2% 40.0% (0/14) (8/14) (0/15) (6/15) (7/14) (9/13) (6/15) 12Weeks^(a) 7.1% 50.0% 6.7% 60.0% 35.7% 61.5% 40.0% <0.001 (1/14) (7/14)(1/15) (9/15) (5/14) (8/13) (6/15) 14 Weeks 0.0% 57.1% 6.7% 40.0% 35.7%61.5% 40.0% (0/14) (8/14) (1/15) (6/15) (5/14) (8/13) (6/15) 16Weeks^(a) 0.0% 64.3% 6.7% 60.0% 50.0% 53.8% 40.0% <0.001 (0/14) (9/14)(1/15) (9/15) (7/14) (7/13) (6/15) 18 Weeks 7.1% 50.0% 6.7% 71.4% 46.2%61.5% 57.1% (1/14) (7/14) (1/15) (10/14) (6/13) (8/13) (8/14) 20 Weeks7.1% 53.8% 0.0% 53.3% 35.7% 46.2% 40.0% (1/14) (7/13) (0/15) (8/15)(5/14) (6/13) (6/15) 22 Weeks 0.0% 38.5% 0.0% 46.7% 14.3% 53.8% 26.7%(0/14) (5/13) (0/15) (7/15) (2/14) (7/13) (4/15) 26 Weeks^(a) 0.0% 21.4%6.7% 40.07% 14.3% 46.2% 20.0% 0.008 (0/14) (3/14) (1/15) (6/15) (2/14)(6/13) (3/15)^(a)Evaluation visits pre-specified for analysis. The response rates atPaulus 50% are shown in Table 4. The magnitude of the clinical benefitof cA2 treatment was substantial. The majority of patients wereresponding to cA2 treatment according to the 50% Paulus criteria.

Commensurate with the clinical response rates shown in Tables 2-4, mostof the patients in the treatment groups demonstrating effectiveness ofcA2 treatment received all 5 infusions of cA2 (Table 5). The principlereason for patients not receiving the complete dose regimen was becauseof lack of efficacy in the placebo group (methotrexate alone) and in the1 mg/kg group not receiving methotrexate. All 15 patients in the 3 mg/kggroup that received methotrexate completed the 5-infusion dose regimen.TABLE 5 Number of Infusions Completed Treatment Groups Pts with Placebo1 mg/kg cA2 3 mg/kg cA2 10 mg/kg cA2 Treatment complete^(a) MTX+ MTX+MTX− MTX+ MTX− MTX+ MTX− effect infusions (n = 14) (n = 14) (n = 15) (n= 15) (n = 14) (n = 14) (n = 15) p-value 5 infusions 6 12  8 15  12  12 12  0.003 (42.86%) (85.71%) (53.33%) (100.00%) (85.71%) (85.71%)(80.00%) 4 infusions 0 1 0 0 1 1 0 (0.00%) (7.14%) (0.00%) (0.00%)(7.14%) (7.14%) (0.00%) 3 infusions 2 1 6 0 0 1 1 (14.29%) (7.14%)(40.00%) (0.00%) (0.00%) (7.14%) (6.67%) 2 infusions 5 0 1 0 1 0 2(35.71%) (0.00%) (6.67%) (0.00%) (7.14%) (0.00%) (13.33%) 1 infusion 1 0.0 0 0 0 0 (7.14%) (0.00%) (0.00%) (0.00%) (0.00%) (0.00%) (0.00%)^(a)Patients are counted only once for the first group for which theyqualify (5 infusions >4 infusions etc. . . ). Patients were only countedif they had completed the entire infusion.

Results for measures of swollen and tender joint counts and thephysician and patient global assessments are shown in FIGS. 1-4. Themedian results in FIGS. 1-4 were reported for each evaluation visitbased only on the patients with data collected. That is, a lastobservation carried forward approach was not used for patients whodropped out. Instead, the number of patients with data that compriseeach point on the graph was reported at the bottom of the figures.

Despite the number of drop-outs in the placebo group and the 1 mg/kggroup not receiving methotrexate, the results in FIGS. 1-4 demonstratethat cA2 treatment in combination with methotrexate profoundly reducesdisease activity for all of the traditional measurements of diseaseactivity, approaching near remission in many patients.

Results for a commonly used serum marker of inflammatory activity,C-reactive protein (CRP) are shown in FIG. 5. Treatment with cA2produced a rapid decrease in CRP concentration which was sustainedthrough 26 weeks in the patients who received 3 or 10 mg/kg cA2.

Results for the Health Assessment Questionnaire (HAQ) are shown in FIG.6. This measurement of quality of life/disability demonstratedimprovement over time corresponding with the clinical improvementobserved in patients treated with cA2. In the patients treated with 3mg/kg cA2 and methotrexate, the HAQ decreased from 2.0 at baseline to1.1 at 22 weeks.

Pharmacokinetics of cA2

Serum concentrations of cA2 were obtained in all patients in this study.The serum concentration in each patient plotted over time according tothe cA2 dose group is shown in FIG. 7. Data plotted are the serum cA2concentrations obtained just before the administration of cA2 at weeks2, 6, 10 and 14 and then at weeks 18 and 26. These sampling times wereselected to best demonstrate the stability of the cA2 concentrationduring the multiple dose regimen and the decline in serum cA2concentration after the last dose was administered. For purposes of datapresentation, the scales for cA2 concentration for each graph arecondensed as the cA2 dose was increased.

Substantial differences were observed for the cA2 serum concentrationover time in the 1 mg/kg dose groups according to whether patientsreceived methotrexate. Most of the patients receiving 1 mg/kg cA2 withmethotrexate demonstrated measurable cA2 concentrations through 18weeks, although it appeared that there was a tendency for theconcentration to decline over time. In sharp contrast, the majority ofpatients who received 1 mg/kg cA2 without methotrexate were not able tomaintain measurable serum concentrations of cA2 over time. As discussedherein, the inability to maintain serum cA2 in these patients wasassociated with a high rate of neutralizing antibody formation.

In contrast to the 1 mg/kg groups, patients who received either 3 mg/kgcA2 or 10 mg/kg cA2 were able to maintain serum cA2 concentrationsthrough the multiple dose regimen. However, even in those dose groups,there was evidence that concomitant treatment with methotrexate wasassociated with high cA2 serum concentrations. As shown in FIG. 8, themedian serum cA2 concentration in both the 3 and 10 mg/kg dose groupsreceiving methotrexate was higher than in the corresponding groups notreceiving methotrexate.

Immune Responses to cA2

Serum samples were collected through 26 weeks from all patients andanalyzed for human anti-chimeric antibodies (HACA) to cA2. The resultsfor HACA responses for each cA2 treatment group are shown in Table 6. Itshould be noted that in several patients in the 3 mg/kg group and inmost patients in the 10 mg/1 g group, cA2 was still present in the26-week sample and could potentially interfere with the detection ofHACA in the assay. However, it could also be reasoned that ifneutralizing antibodies were present at 26 weeks, then cA2 should not bepresent. Therefore, in presenting the data in Table 6, results for theimmune response rate are shown not including patients with serum cA2 at26 weeks and including patients with serum cA2 at 26 weeks, assumingthat if cA2 was present at 26 weeks, the patient did not have a positiveHACA response. TABLE 6 HACA Responses 1 mg/kg 3 mg/kg 10 mg/kg MTX+ MTX−MTX+ MTX− MTX+ MTX− HACA 2/13 8/15 0/10 3/12 0/2 1/10 responses not(15.4%) (53.3%) (0%) (25.0%) (0%) (10%) including pts with 26-week serumcA2 HACA 2/13 8/15 0/15 3/14 0/14 1/15 responses (15.4%) (53.3%) (0%)(21.4%) (0%) (6.7%) including pts with 26-week serum cA2¹¹Patients with a measurable 26-week serum cA2 concentration wereconsidered negative for a HACA response for this analysis.

The results in Table 6 demonstrate that concomitant methotrexatetreatment suppresses the immune response to cA2, enabling stablepharmacokinetics to be achieved in a multiple dose regimen of cA2. Thiseffect was also found after combined anti-CD4/anti-TNF antibodytreatment in mice with collagen-induced arthritis and described in U.S.application Ser. No. 08/607,419, filed Feb. 28, 1996, the teachings ofwhich are entirely incorporated herein by reference.

Clinical Safety

Two out of 86 patients (with most patients receiving 5 treatments)experienced multisystem infusion-related reactions with retreatment.Multisystem, infusion-related reactions include headache, fever, facialflushing, pruritus, myalgia, nausea, chest tightness, dyspnea, vomiting,erythema, abdominal discomfort, diaphoresis, shivers, hypertension,lightheadedness, hypotension, palpitations and somnolence.

Hypersensitivity reactions, as described herein, may occur wheneverprotein-containing materials, such as cA2, are administered. Thus, it isunclear whether these symptoms represent an immunologic event orphysical factors such as infusion rate and immunoglobulin aggregation.Investigators have reported that symptoms resolve in some patients bydecreasing the rate of the infusion. Previous literature reportsindicate that vasomotor symptoms have been observed in patientsreceiving intravenous immunoglobulin therapy (Berkman et al., Ann.Intern Med. 112:278-292 (1990); Ochs et al., Lancet 2:1158-1159 (1980)).

One patient developed hypotension during all three infusions of 10 mg/kgcA2. The patient did not display clinical signs of hypotension and didnot require medical treatment, but, in keeping with predefined safetycriteria, the treatment schedule of this patient was discontinued.

One patient treated with 3 infusions of 10 mg/kg of cA2 and with 7.5mg/week methotrexate developed symptoms of sepsis as a result ofstaphylococcal pneumonia 2 weeks after her last study visit and 14 weeksafter her last infusion with cA2. Six days after developing symptoms shewas admitted to the hospital and treated. She died one day later. (Thispatient had not proceeded with the fourth infusion for reasons unrelatedto the sepsis.) Patients with RA who develop infections have a worsethan expected outcome. Wolfe and coworkers have reported anobserved:expected ratio for death due to pneumonia of 5.307 and anobserved:expected ratio for death due to infections (excludingpneumonia) of 6.213 in RA patients from the ARAMIS database (Wolfe etal., Arthritis Rheumatism 4:481-494 (1994)).

One patient experienced a serious postoperative infection followingcataract surgery 9 weeks after the fifth and last infusion of 3 mg/kg ofcA2 (with 7.5 mg/week methotrexate), leading to removal of the eye. Thispatient was receiving prednisolone (7 mg/day). The incidence ofendophthalmitis after cataract extraction has been reported to bebetween 0.072 and 0.093% (Kattan et al., Ophthalmology 98(9): 1147-1148(1991)) and may be heightened in patients receiving corticosteroidtherapy.

Eight (9%) of 87 patients developed double stranded (ds)-DNA antibodiesfollowing multiple infusions of cA2. Measurements were performed atbaseline, week 8, 16 and 26 (12 weeks following the last infusion). Inthese patients with antibodies against ds-DNA, there was a trend towarda lower level in antibodies at the last evaluation, with two patientsbeing negative.

One patient developed dyspnea, pleuritic chest pain and a rebound ofarthritis activity at study week 14 (four weeks after the fourthinfusion of 3 mg/kg of cA2). Symptoms resolved and she received herfifth dose of cA2. Symptoms recurred 3 weeks later. Examination of theserial blood samples revealed that the test for antinuclear antibodiesand anti ds-DNA antibodies were negative prior to treatment, but becamepositive at week 6 of the study. The patient's symptoms responded tooral prednisolone 20-30 mg daily. The working diagnosis was systemiclupus erythematosus (SLE). The patient currently does not have symptomsof SLE but has active RA.

To date, although antibodies to ds-DNA have been detected in patientstreated with cA2, they generally represent transient increases and onlyone patient has been symptomatic. In patients who have had sufficientfollow-up, anti-ds-DNA antibodies have resolved with discontinuation oftreatment.

In summary, treatment with cA2 is well tolerated. The reductions indisease activity produced by cA2 are significant as supported by thefindings of a low placebo response rate. High clinical response ratesare obtained with a multiple dose regimen of 3 mg/kg cA2 in combinationwith 7.5 mg/wk methotrexate and can be sustained through 26 weeks. Thisdose regimen is considered preferable to the 1 mg/kg plus methotrexateregimen because better pharmacokinetics are obtained, virtually noimmune response was detected and the clinical response is bettersustained following the last treatment with cA2. The clinical benefitobtained by increasing the dose regimen to 10 mg/kg cA2 plusmethotrexate is similar to that observed with the 3 mg/kg cA2 plusmethotrexate regimen.

Thus, the results of this study indicate that treatment with a multipledose regimen of cA2 as adjunctive and/or concomitant therapy tomethotrexate therapy, in RA patients whose disease is incompletelycontrolled by methotrexate, produces a highly beneficial or synergisticclinical response that can be sustained through 26 weeks. The benefitproduced by cA2 generally exceeds 50% reductions in the traditionalmeasurements of rheumatoid arthritis (swollen and tender joints, patientand physician global disease assessments) and achieves near clinicalremission in many patients. Accordingly, the results of this studyindicate that treatment with multiple infusions of cA2 as adjunctiveand/or concomitant therapy to methotrexate therapy is an important andefficacious therapeutic approach for treating RA in patients.

Example 2 Clinical Treatment of Rheumatoid Arthritis by Single Infusionof an Anti-TNF Antibody in Patients Receiving Methotrexate

A randomized, double-blind, placebo controlled study was conducted toevaluate the effects of a single infusion of placebo, 5, 10 or 20 mg/logcA2 in combination with methotrexate, administered at a dose of 10mg/week, in the treatment of rheumatoid arthritis (RA) in patients.

Patients

Twenty-eight (28) RA patients at three centers in the United States who,despite receiving three months therapy with methotrexate administered ata stable dose of 10 mg/wk for at least 4 weeks prior to screening, stillhad active disease according to the criteria of the American College ofRheumatology, were enrolled in the study. Active disease was defined bythe presence of six or more swollen joints plus at least three of foursecondary criteria (duration of morning stiffness ≧45 minutes; ≧6 tenderor painful joints; erythrocyte sedimentation rate (ESR) ≧28 mm/hour;C-reactive protein (CRP) ≧20 mg/l.

Patients taking NSAIDs and corticosteroids (prednisone) at screeningwere allowed to continue at stable doses (7.5 mg/day).

Study Infusions

The chimeric monoclonal anti-TNF antibody (cA2) was supplied as asterile solution containing 5 mg cA2 per ml of 0.01 M phosphate-bufferedsaline in 0.15 M sodium chloride with 0.01% polysorbate 80, pH 7.2. Theplacebo vials contained 0.1% human serum albumin in the same buffer.Before use, the appropriate amount of cA2 or placebo was diluted to 300ml in sterile saline by the pharmacist, and administered intravenouslyvia a 0.2 μm in-line filter over 2 hours. The characteristics of theplacebo and cA2 infusion bags were identical, and the investigators andpatients did not know which infusion was being administered.

Assessments

Patients were randomized to one of four treatment groups (7 patients pergroup). Each of the 28 patients received a single dose of either 0, 5,10 or 20 mg/kg cA2 and were followed for 12 weeks. Patients continuedtreatment with methotrexate (Pharmacochemie, Netherlands) administeredat 10 mg/week throughout the study. Patients were monitored for adverseevents during infusions and regularly thereafter, by interviews,physical examination, and laboratory testing.

The primary measurement of clinical response was defined by the ACRpreliminary definition of response (Felson et al., Arthritis Rheumatism38(6):727-735 (1995)). Patients were considered to have a response ifthey had a 20% reduction in swollen and tender joint count, and hadexperienced a 20% reduction in 3 of the 5 following assessments:patient's assessment of pain (VAS), patient's global assessment ofdisease activity (VAS), physician's global assessment of diseaseactivity (VAS), patient's assessment of physical function (HAQ), and anacute phase reactant (ESR). The ESR was measured at each study site witha standard method (Westergen).

Evaluations were performed at day 3, and at weeks 1, 2, 4, 6, 8, 10, and12.

Results

The 28 patients were randomized to one of four treatment (or dose)groups.

The clinical response rates over time by ACR 20% criteria in each of thetreatment groups is shown in Table 7. TABLE 7 Clinical Response Rates(By ACR 20% Criteria) In Patients Receiving 10 mg/kg Methotrexate cA2Dose of cA2 Treated Placebo 5 mg/kg 10 mg/kg 20 mg/kg Patients Ptsevaluated 7 7 7 7 21 Pts with 1(14.3%) 6(85.7%) 5(71.4%) 6(85.7%)17(81.0%) any response 1 Week 0(0.0%) 4(57.1%) 2(28.6%) 5(71.4%)11(52.4%) 2 Weeks 0(0.0%) 4(57.1%) 5(71.4%) 5(71.4%) 14(66.7%) 4 Weeks1(14.3%) 3(42.9%) 5(71.4%) 5(71.4%) 13(61.9%) 6 Weeks 0(0.0%) 3(42.9%)5(71.4%) 4(57.1%) 12(57.1%) 8 Weeks 1(14.3%) 3(42.9%) 4(57.1%) 4(57.1%)11(52.4%) 10 Weeks 1(14.3%) 1(14.3%) 4(57.1%) 3(42.9%) 8(38.1%) 12 Weeks1(14.3%) 2(28.6%) 4(57.1%) 3(42.9%) 9(42.9%)

Clinical benefit of cA2 treatment was evident at the first evaluationvisit at one week. Although each of the 3 doses of cA2 produced clinicalresponses in the majority of patients treated, the duration of clinicalresponse appeared to be better sustained through 12 weeks in the groupsreceiving 10 or 20 mg/kg cA2. Clinical response was achieved much morefrequently among patients receiving cA2 as compared to placebo. That is,17/21 (81%) patients in the 3 cA2 groups achieved a response, comparedwith only 1/7 (14%) placebo treated patients. The magnitude of clinicalresponse was notable. The mean tender joint count among cA2 treatedpatients decreased from 30.1 at baseline to 13.3 at week 12, and meanCRP decreased from 3.0 at baseline to 1.1 at week 12.

The duration of clinical response appeared to be dose dependent. 2/6(33%) of the responding patients treated with 5 mg/kg cA2 sustained aresponse through 12 weeks of followup, compared to 7/11 (64%) of theresponding patients who received 10 or 20 mg/kg. Treatment in all groupswas generally well tolerated.

In summary, the results of this study indicate that treatment with cA2as adjunctive and/or concomitant therapy to methotrexate therapy iseffective in the reduction of the signs and symptoms of rheumatoidarthritis in patients whose disease is incompletely controlled bymethotrexate. Moreover, the clinical response achieved by this approachcan be sustained for more than 12 weeks after a single treatment.Accordingly, the results of this study indicate that treatment with cA2as adjunctive and/or concomitant therapy to methotrexate therapy is animportant and efficacious therapeutic approach for treating RA inpatients.

Example 3 Clinical Treatment of Rheumatoid Arthritis by Repeated DoseAdministration of an Anti-TNF Antibody in Patients Following a SingleDose, Double-Blind, Placebo-Controlled Trial

An open label study was conducted to evaluate the effects of repeatedinfusions of 10 mg/kg cA2 in combination with methotrexate, administeredat a dose of 10 mg/week, in the treatment of rheumatoid arthritis inpatients.

Patients

As described in Example 2, a randomized, double-blind, placebocontrolled, 12 week study of cA2 was conducted in RA patients who hadactive disease despite receiving three months therapy with methotrexateadministered at a stable dose of 10 mg/wk for at least 4 weeks prior toscreening.

At week 12, patients who had completed the 12 week evaluation period andhad not experienced adverse events prohibiting further infusions of cA2,were offered 3 subsequent open label infusions of cA2, administered at adose of 10 mg/kg, at eight week intervals (weeks 12, 20, 28).Twenty-three (23) patients from the 12 week study were enrolled in thisstudy.

Assessments

11/23 patients entering this open label study were evaluated at 1 of 3centers in the United States and followed up to 40 weeks after initialentry. Patients continued treatment with methotrexate administered at 10mg/week throughout the study. Repeated treatments with cA2 weregenerally well tolerated. Three patients had transient infusion relatedsymptoms (urticaria, somnolence).

The primary measurement of clinical response was defined by the ACRpreliminary definition of response (Felson et al., Arthritis Rheumatism38(6):727-735 (1995)). Patients were considered to have a response ifthey had a 20% reduction in swollen and tender joint count, and hadexperienced a 20% reduction in 3 of the 5 following assessments:patient's assessment of pain (VAS), patient's global assessment ofdisease activity (VAS), physician's global assessment of diseaseactivity (VAS), patient's assessment of physical function (HAQ), and anacute phase reactant (ESR). The ESR was measured at each study site witha standard method (Westergen).

Results

Of six patients who had all received cA2 during the double-blinded studydescribed in Example 2 and responded through the 12 weeks of that study,four patients sustained a response throughout the 40 week followup. Ofthe remaining two patients, one patient is still responding through week28, and one patient recently entered this open label trial. For all 4patients completing 40 weeks of followup and the patient at week 28,final tender joint counts were 2 and swollen joint counts 1, compared toa mean of 23 and 29, respectively, at entry into the double-blindedstudy described in Example 2. For 4 of these 5 patients, ESR were 18mm/hr and CRP 0.7, compared to a mean of 27 and 3.9, respectively, atentry into the double-blind study described in Example 2.

Of two patients who had both received cA2 during the double-blindedstudy described in Example 2 and responded only through week 10 of thatstudy, one patient responded through 36 weeks and one patient is stillresponding through week 20.

Of three patients who did not respond during the double-blinded studydescribed in Example 2 (2 received placebos, 1 received 5 mg/kg cA2),two of these patients experienced a transient clinical response, and onepatient is still responding through week 20.

In summary, the preliminary results of this study suggest that repeatedadjunctive and/or concomitant therapy with cA2, in RA patients whosedisease is incompletely controlled by methotrexate, can result insubstantial clinical improvement for a majority of the patients.Moreover, the clinical response achieved by this approach can besustained for up to 40 weeks of followup. Accordingly, the results ofthis study indicate that repeated treatment with cA2 as adjunctiveand/or concomitant therapy to methotrexate therapy is an important andefficacious therapeutic approach for treating RA in patients.

EQUIVALENTS

Those skilled in the art will know, or be able to ascertain, using nomore than routine experimentation, many equivalents to the specificembodiments of the invention described herein. These and all otherequivalents are intended to be encompassed by the following claims.

1. A method for treating or preventing a tumor necrosis factor-mediateddisease in an individual in need thereof comprising co-administeringmethotrexate and a TNFα antagonist to said individual, intherapeutically effective amounts. 2-38. (canceled)